We propose to identify and develop small molecule inhibitors that target two previously neglected stages of the HIV life cycle: virus budding and capsid assembly. In the first aim, we will focus on developing inhibitors of the essential interaction between the cellular protein Tsgl01 and the p6 domain of the HIV-1 Gag protein. In preliminary studies, we have discovered that human Tsgl01 performs an essential role in virus budding by binding to the p6 domain of the HIV Gag protein and recruiting other cellular factors in the Vps pathway that facilitate virus release. We have also determined the solution structure of the p6-binding UEV domain of Tsgl 01 . Our collaborators at Myriad Pharmaceuticals have discovered lead compounds that inhibit the Tsgl01 UEV/p6 interaction. We will now determine the structure of Tsgl01 UEV in complex with its p6 binding site and in complex with Myriad's inhibitors as part of a comprehensive program in structure-assisted development of inhibitors of HIV budding. In the second aim, we will focus on discovering small molecule inhibitors of HIV capsid assembly. In principle, viral capsid assembly represents an attractive target for therapeutic intervention, but the lack of suitable assays for discovering lead compounds has been a major impediment in this research. In preliminary studies, we have determined structures of the HIV CA protein in two different conformational states, characterized the higher order assemblies formed by recombinant CA proteins, and overcome a number of hurdles in inhibitor assay development. We now propose to pursue the discovery of HIV capsid assembly inhibitors by adapting our assays to run on high-throughput platforms, screening chemical libraries for small molecule inhibitors, and characterizing the biochemistry and structural biology of the lead compounds that emerge from our screens.